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REVIEW ARTICLE OPEN Regulation of RIG-I-like receptor-mediated signaling: interaction between and viral factors

Koji Onomoto1, Kazuhide Onoguchi1 and Mitsutoshi Yoneyama 1

Retinoic acid-inducible I (RIG-I)-like receptors (RLRs) are RNA sensor molecules that play essential roles in innate antiviral immunity. Among the three RLRs encoded by the human , RIG-I and melanoma differentiation-associated gene 5, which contain N-terminal caspase recruitment domains, are activated upon the detection of viral in the of -infected cells. Activated RLRs induce downstream signaling via their interactions with mitochondrial antiviral signaling and activate the production of type I and III and inflammatory cytokines. Recent studies have shown that RLR-mediated signaling is regulated by interactions with endogenous RNAs and host proteins, such as those involved in stress responses and posttranslational modifications. Since RLR-mediated cytokine production is also involved in the regulation of acquired immunity, the deregulation of RLR-mediated signaling is associated with autoimmune and autoinflammatory disorders. Moreover, RLR- mediated signaling might be involved in the aberrant cytokine production observed in coronavirus disease 2019. Since the discovery of RLRs in 2004, significant progress has been made in understanding the mechanisms underlying the activation and regulation of RLR-mediated signaling pathways. Here, we review the recent advances in the understanding of regulated RNA recognition and signal activation by RLRs, focusing on the interactions between various host and viral factors.

1234567890();,: Keywords: RIG-I-like receptors; Viral infection; Innate immunity; Stress response

Cellular & Molecular Immunology (2021) 18:539–555; https://doi.org/10.1038/s41423-020-00602-7

INTRODUCTION Type III IFNs, similar to type I IFNs, are also activated in response Interferons (IFNs), the family of cytokines first discovered, were to pathogenic infections; however, these cytokines are recognized identified as host factors secreted by virus-infected cells to by a distinct receptor complex comprising IFNLR1 and interleukin “interfere” with .1,2 Based on the subsequent (IL)-10R2.5 The signaling pathway downstream of the type III IFN identification of their corresponding , they were further receptor complex is nearly identical to that of type I IFNs, resulting classified into three types in humans: type I IFNs [multiple IFN-α in similar antiviral activity via JAK-STAT-induced ISG production.3 forms (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21), IFN-β,IFN-ε,IFN-κ, The functional differences between type I and type III IFNs might and IFN-ω], type II IFN (a single form, IFN-γ), and type III IFNs be caused by the different types producing IFNs and their (IFNλ1-4).3 Among these types, type I and type III IFNs play essential cognate receptors.5 Notably, type III IFN receptors are predomi- roles in innate antiviral immunity. Infections caused by microbes, nantly expressed on epithelial cells and certain myeloid cells, including and bacteria, transiently induce the secretion of suggesting their critical roles in these cell types.5 type I IFNs, which associate with the type I IFN receptor complex IFNs have been used for the treatment of several viral infections (IFNAR1 and IFNAR2) expressed by the infected cells themselves and various immunological disorders and due to their and surrounding noninfected cells, thereby activating cytoplasmic antiviral effects and cell growth inhibitory properties.6 Although Janus kinases (JAK1 and TYK2). Signal transducer and activator of type I IFNs have previously comprised the sole treatment strategy transcription (STAT1) and STAT2, activated by JAK-mediated for virus (HCV), they have more recently been replaced tyrosine phosphorylation, together with the DNA-binding compo- by nonstructural (NS)3/4A and NS5A inhibitors, which nent IFN regulatory factor (IRF)-9, form a tripartite complex, referred have proven effective for the complete elimination of HCV. IFN to as IFN-stimulated gene factor 3 (ISGF3). ISGF3 then is therapy has been proposed as a potential strategy for the translocated to the nucleus, where it binds to the IFN-stimulated treatment of coronavirus disease 2019 (COVID-19) caused by response element (ISRE) on the promoters of hundreds of IFN- severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).7 inducible genes (ISGs) and activates their transcription. Proteins Indeed, models of SARS-CoV, another pathogenic corona- encoded by ISGs induce strong antiviral innate immunity to virus, have exhibited delayed induction of type I IFNs during the eliminate invading viruses.4 Since the major histocompatibility early onset of infection, leading to potential excessive cytokine complex (MHC) and costimulatory molecules are also ISGs, IFNs secretion, called a “”, which is associated with play critical roles in regulating acquired immunity (Fig. 1). exacerbated disease.8 This outcome is also observed in COVID-19,

1Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8673, Japan Correspondence: Mitsutoshi Yoneyama ([email protected]) These authors contributed equally: Koji Onomoto, Kazuhide Onoguchi Received: 30 September 2020 Accepted: 17 November 2020 Published online: 18 January 2021

© The Author(s) 2021 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 540 cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor.14 The transcription factors IRF-3/7 and nuclear factor-κB (NF-κB), downstream of IFN-inducing PRRs, are commonly activated and transiently induce IFN-encoding genes. Genes encoding proin- flammatory cytokines, such as IL-6 and tumor necrosis factor (TNF)-α, are also induced by activated NF-κB. In this review, we focus on RLRs that detect cytoplasmic viral RNAs and the molecular mechanisms underlying RNA-mediated activation of RLRs and signaling regulation performed by host and viral factors.

ACTIVATION OF RLR-MEDIATED SIGNALING Association between RLRs and MAVS RIG-I, which enhances IFN production in response to RNA virus infection, was isolated via expression cloning.15 The encodes three RLRs, RIG-I, melanoma differentiation- associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), which are DExD/H box-containing RNA and ubiquitously expressed in the cytoplasm.16 These RLRs commonly have a central domain and a C-terminal domain (CTD) critical for RNA recognition. RIG-I and MDA5 also contain a tandem caspase recruitment domain (CARD) in their N- terminal region, which is required for their association with the adapter molecule mitochondrial antiviral-signaling protein (MAVS; Fig. 1 -mediated antiviral responses and diseases. In an also known as IPS-1, VISA, and Cardif), which also contains a single antiviral , sensors detect viral CARD at its N-terminus (Fig. 2a). The interaction between the nucleic acids intracellularly and extracellularly, which then activate CARDs of RIG-I/MDA5 and MAVS leads to the recruitment of intracellular signaling pathways, leading to transcriptional activation downstream signaling molecules, including TNF receptor- of type I and type III interferon (IFN) genes upon the nuclear associated factor (TRAF)3/6 and inhibitor of NF-κB kinase (IKK) translocation of the transcription activators IFN regulatory factor family members (IKKε, TBK1, and IKKα/β), to activate IRF-3/7 and (IRF)-3/7 and nuclear factor-κB (NF-κB). Secreted type I and type III NF-κB, leading to the transcriptional activation of IFN and IFNs activate a secondary signaling pathway by binding to their fl 17,18 cognate IFN receptor (IFNR) complexes in an autocrine and proin ammatory cytokine genes (Fig. 2b). More importantly, paracrine manner. The downstream JAK-STAT pathway activates since RLR genes are ISGs, a positive feedback loop leads to further the transcription complex IFN-stimulated gene factor 3 (ISGF3), enhancement of IFN production.15 which leads to the expression of hundreds of IFN-stimulated genes RIG-I detects substrate RNAs that have a panhandle double- (ISGs), resulting in strong antiviral responses. Since IFN-induced stranded structure with a 5′-triphosphate or 5′-diphosphate antiviral activity is critical for eliminating infected viruses, its weak or moiety and activates downstream signaling in an ATP- delayed response leads to uncontrolled infectious disease. In dependent manner (Fig. 2b).19 A gene knockout (KO) study in contrast, IFNs also play essential roles in regulating the subsequently mice revealed that RIG-I can be used for detecting multiple viral induced adaptive immunity, and thus, their excessive or chronic 20 activation can lead to the development of autoimmune or in many cell types. The study showed that RIG-I KO in autoinflammatory diseases as, well as the initiation of cytokine mice results in embryonic lethality resulting from liver degenera- storm, which exacerbates inflammation tion; however, another study showed that KO mice with a different genomic region targeted were viable but developed spontaneous colitis.21 Although the mechanism underlying colitis development suggesting a potential therapeutic strategy to enhance the initial in these mice has not been fully elucidated, recent studies have production of IFNs during the early stages of SARS-CoV-2 demonstrated that RIG-I might participate in the maintenance of infection.9 However, excessive IFN production in the late stages homeostasis in digestive organs by interacting with commensal of infection is associated with the development of cytokine storm, microorganisms.22,23 suggesting that the therapeutic use of IFNs requires careful MDA5 recognizes relatively long dsRNAs.24 Specifically, studies consideration in terms of timing and dosage.10,11 The use of type with MDA5-KO mice have demonstrated that MDA5 is primarily III IFNs in COVID-19 treatment has also been suggested due to involved in detecting viruses belonging to the Picornaviridae their limited action on cell types such as epithelial cells;7 however, family;25 however, it can also recognize several other viral species. recent reports have shown that they may be detrimental to In addition to their virus-sensing roles, RIG-I/MDA5 are both COVID-19 patients,12,13 and thus, further investigations into their capable of controlling viral replication by exhibiting an “effector- suitability as therapeutic agents are warranted. like” function that competitively block the interaction between In higher , pathogenic infections are detected by viral proteins and RNA.26–28 pattern recognition receptors (PRRs), which are part of the innate Another RLR, LGP2, does not harbor N-terminal CARD and immune system. Toll-like receptors (TLRs) and C-type lectin therefore does not function as a signaling molecule. Although receptors (CLRs) are extracellular PRRs that detect pathogens on in vitro studies, including our own, suggested that LGP2 acts as a the plasma membrane or endosomal compartment. In addition, negative regulator of RIG-I/MDA5-mediated signaling,16,29,30 and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), Nod-like subsequent studies with KO mice revealed a positive regulatory receptors (NLRs), and cytoplasmic DNA sensors serve as intracel- function for LGP2.31,32 These reports revealed that LGP2 deficiency lular PRRs. The production of type I and type III IFNs is explicitly impairs IFN production in response to , including mediated by PRRs, such as endosomal TLRs and RLRs that detect encephalomyocarditis virus (EMCV), suggesting that LGP2 is nucleic acids. Among these PRRs, TLR-3 recognizes double- involved in the MDA5-mediated signaling activation (the mole- stranded RNA (dsRNA), TLR-7/8 recognizes single-stranded RNA cular mechanisms are described as follows). In contrast, the (ssRNA), and TLR-9 recognizes unmethylated CpG DNA in the physiological significance of LGP2 in RIG-I-mediated signaling endosomal compartment. RLRs recognize cytosolic RNAs, and remains unclear because LGP2 deficiency has different effects

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 541

Fig. 2 RIG-I-like receptors and the associated activation machinery. a Primary structure of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and mitochondrial antiviral-signaling protein (MAVS). All RLR family members contain a DExD/H box-containing RNA helicase domain composed of Hel1, Hel2i, Hel2, and pincer domains and the C-terminal domain (CTD). RIG-I and melanoma differentiation-associated gene 5 (MDA5), but not laboratory of genetics and physiology 2 (LGP2), contain repeated caspase recruitment domains (CARDs) in their N-terminal regions. MAVS has an N-terminal single CARD, three TNF receptor-associated factor (TRAF)-binding motifs (TBMs), and a transmembrane (TM) domain in the C-terminus. b RIG-I and MDA5 recognize 5′-triphosphate(ppp)-containing panhandle double-stranded (ds)RNA and long dsRNA, respectively. Recognition of substrate RNAs induces ATP-dependent conformational changes of RLRs, which induce filamentous oligomer formation on substrate RNAs. The N-terminal CARDs adopt a “lock-washer”-like tetramer with K63-linked ubiquitin chains and form signal-competent oligomers with the CARD of MAVS, which is expressed on the outer membrane of mitochondria. MAVS oligomers recruit downstream signaling molecules, such as TRAFs and inhibitors of NF-κB kinases (IKKs), which leads to the activation of IRF-3/7 and NF-κB depending on the viral infection.31,32 LGP2 has also been reported Activation of RLR-mediated signaling to regulate RNA silencing machinery. Specifically, it can associate In the steady state, RIG-I is likely to adopt an autorepressed with , an for microRNA (miRNA) produc- conformation via direct interactions between CARDs and the tion, suggesting that LGP2 might indirectly regulate antiviral helical insertion domain (Hel2i) located in the helicase domains.44 signaling by regulating .33 Moreover, LGP2 has An initial structural analysis of RIG-I CARD showed that the CTD been reported to interact with transactivation response RNA- with substrate dsRNA forms a rigid -resistant structure, binding protein (TRBP), a positive regulator of Dicer-mediated indicating a critical role of the CTD in substrate recognition.45,46 miRNA maturation, to inhibit the production of several miRNAs The positively charged surface of the RIG-I CTD was also shown to and induce the apoptotic pathway by enhancing the expression of be critical for recognizing the 5′-triphosphate signature of specific genes.34,35 In the case of (WNV) infection, substrate dsRNA. Subsequent structural analysis of the helicase LGP2 plays an essential role in the survival and adaptation of domain and CTD of RIG-I with substrate RNA revealed that both CD8+ T cells but not in MAVS-mediated IFN production.36 Taken wrap around dsRNA with CTD sheathing the end.44,47–49 The together, these observations suggest that LGP2 might be involved interaction between RIG-I and its dsRNA substrate induces an ATP- in a broad range of antiviral effects, and hence, further analysis is dependent intramolecular conformational change and the release required. of N-terminal CARDs, allowing the RIG-I CARD to associate with the MAVS is an adapter molecule for RLR-mediated signaling that MAVS CARD on the mitochondria. At the same time, the contains an N-terminal CARD and C-terminal transmembrane (TM) activity of RIG-I allows it to migrate along the RNA domain required for its localization to intracellular membranes, substrate; thus, the accumulation of multiple RIG-I molecules on including those of mitochondria, and three TRAF-binding motifs RNA results in the formation of filamentous oligomers.50–52 (TBMs) required for signal transduction. A MAVS-KO mouse model Furthermore, the CARDs of the activated RIG-I proteins on the was used to clearly identify the essential role of MAVS in RIG-I/ RNA form a helical tetrameric “lock washer” structure, allowing the MDA5-mediated IFN production.37 MAVS CARD to form signal-competent aggregates (Fig. 2b).53 Several studies have suggested that RLRs/MAVS are involved in In contrast, the constitutive activity of MDA5 is repressed by inflammasome-mediated proinflammatory cytokine production, phosphorylation.54 In contrast to RIG-I, the MDA5 CTD forms a such as IL-1β and IL-18.38 RIG-I has been reported to be directly relatively open structure, indicating that the MDA5 CTD cannot associated with apoptosis-associated speck-like protein containing function independently to recognize RNA.55 A structural analysis of CARD (ASC) and caspase-1 of the inflammasome to form virus- MDA5 with substrate dsRNA demonstrated that the helicase specificinflammasomes in response to vesicular stomatitis virus domain and CTD of MDA5 recognize internal duplexes rather than (VSV) and influenza A virus (IAV) infection.39,40 MAVS is also theendofthedsRNAsubstratestructures.49 Moreover, MDA5 reportedly involved in the formation of the NLRP3 inflammasome molecules accumulate on long dsRNA in a head-to-tail arrangement, in mitochondria.41,42 Furthermore, MAVS-mediated signaling is forming a filamentous structure and transmitting a signal by the N- required to increase permeability and the terminal CARDs protruding from the filaments.49,56 Furthermore, the subsequent K+ ion efflux that leads to the formation of the formation of the MDA5 filament is essential for distinguishing NLRP3-containing inflammasome.43 Thus, further analysis is between self- and non-self-dsRNA via assembly/disassembly warranted to better understand virus-induced inflammasomes. dynamics that depend on the length of the substrate dsRNA.57,58

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Fig. 3 Regulation of RLR-mediated signal transduction. A summary of the regulatory mechanisms of RLR/MAVS-mediated signaling by host and viral factors. a Activated RLRs interact with MAVS, localize to mitochondria, mitochondria-associated membranes (MAMs), and peroxisomes, where they activate downstream signaling. b RLRs accumulate in virus-induced stress granules (SGs) with viral RNAs, resulting in enhanced signaling. c Several host RNAs can be recognized by RLRs and are involved in the regulation of autoimmunity and . Moreover, RLR/MAVS-mediated signaling is regulated by various host molecules: ubiquitin /deubiquitinases (d), posttransla- tional modifiers (e), protein kinases/ (f), RNA-binding proteins (g), other host proteins (h), and autophagy regulators (i). These molecules are classified in square boxes (orange boxes: positive regulators; blue boxes: negative regulators). The molecules that inhibit or promote these regulatory molecules are shown on the right side of each column. Regulatory molecules of viral origin are shown in pink rounded rectangles

However, the precise mechanisms by which LGP2 regulates RIG- of RIG-I or MDA5, MAVS CARDs form protease-resistant prion-like I/MDA5-mediated signaling remain unclear. The CTD of LGP2 aggregates on intracellular membranes such as mitochondria. A adopts a structure similar to that of RIG-I and recognizes the blunt structural analysis revealed that tetrameric RIG-I/MDA5 CARDs act end of dsRNA in a 5′-triphosphate-independent manner, reflecting as templates for MAVS CARD filaments to which downstream the differential function of LGP2 in RIG-I- and MDA5-mediated signaling molecules are recruited.65–67 signaling.55,59,60 A structural analysis of full-length LGP2 suggested that it forms a complex with the MDA5-like helicase domain and a Intracellular localization of RLRs and MAVS RIG-I-like CTD.61 LGP2 can augment MDA5-mediated filament In the inactivated state, RLRs are uniformly expressed in the formation.62 Further analysis is required to elucidate the molecular cytoplasm. Since MAVS is expressed on mitochondrial outer machinery involved in the cooperative recognition performed by membranes, the activated RIG-I/MDA5 in filamentous aggregates these RLRs. is located close to mitochondria. MAVS is also localized in Ectopic overexpression of MAVS in vitro causes constitutive IFN mitochondrial-associated endoplasmic reticulum membranes production, suggesting that the activity of MAVS as a signaling (MAMs) and peroxisomes and regulates RLR-mediated signaling adapter is tightly regulated. Although several molecules have (Fig. 3a).68,69 Peroxisomal MAVS is primarily involved in type III IFN been identified as negative regulators of MAVS, their repression induction.70 In addition, a recent study that performed high- does not induce constitutive activation of MAVS, thus suggesting resolution assays reported that microsomal fractions, such as alternative inhibitory mechanisms. Qi et al. reported that the endoplasmic reticulum membranes, but not mitochondria, play expression of multiple N-terminal truncated MAVS isoforms central roles in RLR-mediated signaling.71 Nuclear-localized RIG-I is produced by alternative initiation suppresses the required for detecting IAV, which replicates in the nucleus;72 constitutive activation of full-length MAVS.63 The constitutive however, the mechanism by which it activates MAVS-mediated signaling capacity of full-length MAVS is repressed upon associa- signaling is unclear. Further analysis is required to understand the tion with inactive truncated MAVS isoforms through the TM intracellular localization of RLRs. domain. However, the intramolecular interaction of MAVS Stress granules (SGs) are membrane-less cytoplasmic aggre- suppresses constitutive activation.64 In response to the activation gates to which RLRs are localized, and they can enhance MAVS-

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 543 mediated signaling by acting as platforms for the recognition of exhibited a gain-of-function phenotype similar to that in the foreign viral RNAs by RLRs (Fig. 3b).73 Under various stress A946T model. The mutant mice spontaneously developed conditions, including viral infection, cells produce SGs to inhibit autoinflammatory symptoms, including lupus-like nephritis.112 A cellular protein synthesis and store the protein synthesis more recent study developed a knock-in mouse model with MDA5 machinery (translationally stalled mRNAs and RNA-binding pro- carrying the A946T , and the results showed that it teins) to reinitiate translation after recovery from the stress exacerbated the mouse autoimmune disease through the condition.74 During viral infection, (PKR), a well- upregulation of basal IFN and hypersensitivity to self-RNA.113 known dsRNA-dependent kinase that is also an antiviral ISG, Multiple MDA5 SNPs, in addition to A946T, have also been phosphorylates the eukaryotic translation initiation factor eIF-2α reported to be associated with autoimmune and autoinflamma- and induces the production of SGs.75–78 The inhibition of virus- tory diseases.114,115 Several gain-of-function in IFIH1 are induced and PKR-dependent SG formation significantly impairs also associated with Aicardi-Goutieres syndrome (AGS)116,117 and IFN production when exposed to NS1-defective IAV but not wild- Singleton-Merten syndrome (SMS).118 In addition, a loss-of- type IAV, indicating that IAV-induced SGs are critical for IFN function mutation in IFIH1, Lys365Glu, has been shown to cause response activation and that NS1 can function as a viral inhibitor severe recurrent respiratory viral infections.119 for SG-mediated signaling.73 The accumulation of MAVS-enriched SNPs in DDX58, which encodes RIG-I, are also associated with mitochondria around RIG-I-containing aggregates supports the SMS.120,121 Specifically, Lässig et al. reported that the Glu373Ala hypothesis that SGs serve as platforms for viral detection.79,80 In and Cys268Phe RIG-I mutants found in SMS, which are devoid of the case of Newcastle disease virus (NDV) infection, RIG-I is ATPase activity, interact with endogenous ribosomal RNA and localized to the viral replication complex (vRC) to initiate primary activate unintentional ATP-independent IFN production, resulting IFN induction during the early postinfection phase, and its in SMS development.122,123 However, little is known about the subsequent accumulation in SGs is required for the robust relationship between MAVS and autoimmune diseases, and upregulation of IFN genes, suggesting the importance of although a loss-of-function mutation (Cys79Phe) found in MAVS intracellular dynamics of RIG-I proteins for efficient activation of has been associated with SLE, the molecular mechanism remains the signaling pathway.80 Furthermore, growing evidence suggests unclear.124 that multiple RNA-binding proteins are localized in virus-induced SGs and are involved in IFN production, which strongly suggests an association between SG formation and RLR-mediated signal REGULATION OF RLR-MEDIATED SIGNALING BY RNA enhancement.75,81–87 Moreover, Ras-GTPase-activating protein RECOGNITION SH3-domain-binding protein 1 (G3BP1), which is a critical RNA recognition by RIG-I component of SG formation, is implicated in RLR-mediated An analysis of RIG-I-KO mice revealed that RIG-I is required for the signaling, suggesting a functional interaction between RLRs and detection of RNA viruses, including IAV, NDV, and Sendai virus SG components in innate antiviral responses.88–90 However, (SeV).20 Subsequent analyses revealed that multiple types of RNA several reports have revealed that RLR localization in SGs is not viruses, including HCV,125 WNV,126 (DEV), necessary for IFN production; thus, the requirement for SGs in RLR- (ZIKV),127 hepatitis D virus (HDV),128 hepatitis E virus (HEV),129 and mediated signaling remains unclear.91–94 Although many viruses Hantaan virus (HTNV), can be recognized by RIG-I.130 Since most have evolved strategies to inhibit SG formation, some exploit SG- RNA viruses replicate their own genome using viral-encoded RNA- containing proteins for their proliferation.76,95 Thus, it remains dependent RNA polymerase (RdRP) within the infected cells, RIG-I unclear whether PKR-induced SG formation is directly involved in recognizes viral genomic RNAs based on their 5ʹ-triphosphate the detection of viral RNAs by RLRs.96 To clarify the function of SGs signature.131,132 The double-stranded structures that cannot be in antiviral responses, further analysis of the relationship between unwound by RIG-I helicase activity, and the short panhandle the life cycle of each virus and the requirement of SG formation is blunt-ended structures with 5′-triphosphate ends, are preferential needed. In addition, because SGs are membrane-less aggregates, structures for RIG-I recognition.45,133,134 A pregenomic RNA, which the lack of an appropriate assay system to investigate their has been reverse-transcribed from the genomic DNA of hepatitis B function has been a major problem. However, recent progress virus () genomic DNA, is also recognized by RIG-I.27 based on an in vitro reconstitution system using liquid-liquid Moreover, incoming full-length viral IAV RNAs can be recognized phase separation has enabled an understanding of the molecular by RIG-I.135–137 Since the IAV RNA genome associates with the mechanisms of SG formation.97,98 Future analyses including those virus-derived nucleocapsid protein (NP) and tripartite RdRP using this in vitro system are expected to clarify the physiological complex at the 5′-terminal panhandle structure,138 the mechanism significance of SGs in antiviral responses. by which RIG-I recognizes the protected viral RNA-protein complex (RNP) remains unclear. A recent study showed that RLRs and their involvement in autoimmune and autoinflammatory several host cosensor(s) or regulatory molecule(s) are involved in diseases RIG-I-mediated signaling; however, the molecular machinery Since RLRs play essential roles in the initial production of IFNs and critical for the coordinated detection of viral RNP by RIG-I and in regulating acquired immunity, their aberrant activation leads to its cofactors remains unclear. Interestingly, recent reports have various disorders, including autoimmune and autoinflammatory demonstrated that small RNAs, such as miniviral RNA (mvRNA), diseases.99–101 A genome-wide analysis revealed that single- produced by the aberrant replication of viral RNA via RdRP form nucleotide polymorphisms (SNPs) in IFIH1, which encodes MDA5, aberrant viral RNPs, are preferentially recognized by RIG-I.139,140 In have been associated with these diseases. Smyth et al. demon- the case of SeV infection, RNA with an abnormal panhandle strated that an Ala to Thr mutation at 946 (A946T) of MDA5 structure derived from defective interfering (DI) particles can be (rs1990760) is significantly correlated with the risk of type 1 detected by RIG-I.141 Additionally, the uridine-rich region of the diabetes.102 Subsequent studies demonstrated that the A946T HCV RNA genome can be recognized by RIG-I.125 The short viral mutation is also associated with Graves’ disease,103 rheumatoid RNA suppressor of virus RNA (svRNA), generated by host arthritis,104 multiple sclerosis,105 systemic lupus erythematosus L (RNase L)-mediated cleavage of the HCV RNA (SLE),106–108 selective IgA deficiency,109 and vitiligo.110,111 In genome, is also efficiently detected by RIG-I.142 addition, a functional analysis of A946T-containing MDA5 revealed In addition to the 5′-triphosphate structure, the 5′-diphosphate that this mutant exhibits gain-of-function properties and consti- moiety characteristic of the reovirus (RV) RNA genome can also be tutively activates IFN production.112 Furthermore, Funabiki et al. recognized by RIG-I.143 This substrate specificity for RNA recogni- developed a mouse strain bearing a Glu821Ser mutation that tion by RIG-I clearly shows that the 5′-cap and 5′-monophosphate

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 544 structures of endogenous RNAs, including that of mRNA, tRNA, recognition particle (SRP) component, is upregulated in stromal and rRNA, cannot be recognized by RIG-I as non-self-RNA.144 cells by Notch-Myc signaling from breast cancer cells, and an Schuberth-Wagner et al. further demonstrated that, in addition to excess of 7SL RNA is secreted in exosomes, which in turn activates the 5′-cap structure of the mRNA, 2′-O-methylation at the +1 base RIG-I in breast cancer cells, leading to increased cancer growth, neighbor of the 5′-cap, the N1 position, is critical for evading metastasis, and treatment resistance.161 However, Alu-derived recognition by RIG-I.145 They also showed that the conserved RNA, an endogenous retrotransposon sequence originating from His830 residue of the RIG-I RNA-binding pocket is critical for 7SL RNA, exhibits anticancer effects via RIG-I activation. The distinguishing the 2′-O-methylated RNA at the N1 site, whereas inhibition of heterogeneous nuclear ribonucleoprotein C substituting His830 with Ala enhances IFN production via the (HNRNPC), which is highly expressed in cancer cells, suppresses recognition of 2′-O-methylated endogenous mRNA by RIG-I. HNRNPC-mediated cellular splicing, resulting in the accumulation virus (YFV), belonging to the family, of Alu sequences within the pre-mRNA intron and Alu-mediated encodes a 2′-O-methyltransferase that allows it to escape activation of RIG-I to inhibit cancer growth.162 Several long detection by RIG-I.145 Many viruses have developed this type of noncoding (lnc)RNAs repress the RIG-I pathway. Mouse-specific evasion strategy to avoid detection by RIG-I. For example, viruses lnc-Lsm3b and human-specific lncATV, which are upregulated by belonging to the Picornaviridae family cannot be recognized by viral infection, compete with viral RNAs for detection by RIG-I, RIG-I due to the covalent bonding of the Vpg (viral thereby inhibiting RIG-I-mediated IFN production.163,164 However, protein genome-linked) at the 5′-end of the viral genome.146 involved in posttranscriptional regulation are believed Crimean-Congo hemorrhagic fever virus (CCHFV) and HTNV, to be unrecognized by RIG-I, as they do not contain 5′- belonging to the order; and virus triphosphate and have a 3′-overhang. However, several miRNAs (BDV), belonging to the family, have a 5′-monopho- enriched in uridines are potential RIG-I agonists.165,166 For sphate end that is not recognized by RIG-I.147 A recent study example, miR136, the expression of which is induced by IAV demonstrated that the RNA of human metapneumovirus (HMPV), infection, can inhibit IAV growth by activating RIG-I-mediated belonging to the family, undergoes N6- signals. Moreover, the introduction of exogenous circular RNAs methyladenosine modification to evade recognition by RIG-I.148 (circRNAs) into cells can activate RIG-I in a 5′-triphosphate- or Furthermore, RIG-I can also recognize RNA secreted from double-stranded structure-independent manner.167 Chen et al. bacteria. Specifically, RNA of intracellular pathogenic bacteria, further showed that endogenous circRNAs, generated by back- such as Listeria monocytogenes, which is released into the splicing to covalently link the 3′ and 5′ ends of the RNA exon, cytoplasm, can be detected by RIG-I.149,150 IFN production via avoid RIG-I recognition by exhibiting N6-methyladenosine mod- RIG-I signaling has been observed in response to other ification.168 However, several reports have also demonstrated that intracellular bacteria, including Salmonella enterica serovar Typhi- contaminating short ssRNAs generated during the production of murium151 and Mycobacterium tuberculosis.152 A recent study circRNAs are critical for circRNA-mediated RIG-I activation.169 showed that secreted L. monocytogenes RNA complexed with the Further analysis is required to elucidate the physiological bacterial RNA-binding protein lmo2686/zea can also be recognized significance of circRNA-mediated RIG-I activation. by RIG-I.153 Furthermore, M. tuberculosis RNA is delivered into the Finally, based on accumulating evidence highlighting the extracellular vesicles of macrophages, leading to the activation of physiological significance and molecular machinery of RIG-I- RIG-I-mediated signaling in uninfected cells.154 These observations mediated signaling, research on the agonist/antagonist targeting strongly suggest that RIG-I-mediated signaling has a broad of RIG-I has been initiated. For example, 5′-triphosphate dsRNA function in the self-defense machinery against pathogenic has been shown to exhibit antiviral activity against lethal IAV infection. Notably, further analysis of the maintenance of home- infection.170 Furthermore, a unique RIG-I agonist has been ostasis in the intestinal and respiratory tracts via commensal reported to exhibit significant antitumor activity.171 RIG-I antago- microbes and RIG-I-mediated signaling is necessary.22,23,155 nists have also been identified with the aim of controlling excessive IFN production.172 Interactions between RIG-I and endogenous RNAs Since the 5′-triphosphate signature in host RNAs is apparent RNA recognition by MDA5 during transcription by RNA polymerases, RIG-I can recognize host An analysis of MDA5-KO mice revealed that MDA5 is involved in endogenous RNAs (Fig. 3c). An analysis of IFN production in detecting viruses belonging to the Picornaviridae family, including response to dsDNA showed that 5′-triphosphate RNA transcribed EMCV and Theiler’s encephalomyelitis virus (TMEV).25 A subse- by RNA polymerase III from intracellular poly(dA-dT) DNA can quent study revealed that MDA5 can recognize relatively long induce IFNs via RIG-I.156,157 This III-mediated activation of RIG-I dsRNAs.24 In addition to picornaviruses, MDA5 also recognizes has been analyzed in host responses against DNA virus infections. DEV (Flaviviridae), RV (), a segmented dsRNA virus,173 Next-generation sequencing of RNA samples interacting with RIG-I murine (MNV; ), with Vpg at the 5′-end of the during virus (HSV; ) infection led to viral RNA,174 murine hepatitis virus (MHV) belonging to the the identification of endogenous RNA transcribed from 5S family with a 5′ cap,175 as well as other viruses101. ribosomal RNA pseudogene 141 (RNA5SP141). RNA5SP141, tran- The non-self-RNA signature of these viruses is a double-stranded scribed by RNA pol III in the nucleus, is translocated to the structure generated as a replication intermediate.176 However, the cytoplasm and activates RIG-I-mediated signaling.158 In cells exact RNA structure recognized by MDA5 is unclear, and further infected with Kaposi’s sarcoma-associated herpesvirus (KSHV; analysis is required to determine the specific properties of MDA5 belonging to the Herpesviridae family), an endogenous RNA ligands.177–179 In the case of coronavirus infection, the 2′-O- designated “vault RNA” transcribed by RNA pol III accumulates methylation of viral RNA by 2′-O-methyltransferase encoded by as a 5′-triphosphate-containing RNA due to the reduced expres- viral nonstructural protein 16 (nsp16) suppresses MDA5-mediated sion of DUSP11 (dual-specificity 11) and induces RIG- IFN production;180 however, it is unclear whether 2′-O-methylation I-mediated signaling to inhibit lytic reactivation of KSHV.159 directly impacts the binding of viral RNA to MDA5.145 A recent However, RIG-I can recognize KSHV RNA in a pol III-independent report on MHV showed that the panhandle dsRNA structure manner.160 These findings indicate that activation of the RIG-I containing 5′-polyuridines of negative-sense RNA (PUN RNA) is an pathway via pol III-induced endogenous RNA is a critical host agonist of MDA5.181. Since SARS-CoV-2 belongs to the Coronavir- strategy to fight against DNA virus infections. In addition, idae family as MHV, it might be recognized by MDA5 via a similar endogenous 7SL RNA transcribed by RNA pol III can activate mechanism. Degradation of PUN RNA by MHV nsp15, coding for a RIG-I-mediated signaling in cancer cells. 7SL RNA, a signal polyU-specific endoribonuclease (EndoU), limits MAD5-mediated

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 545 IFN production, suggesting a viral strategy to escape antiviral zinc finger protein, recruits TRIM25 to RIG-I/MDA5 and enhances activity.181 Considering that it is conserved in the SARS-CoV-2 the ubiquitin-mediated activation of RIG-I/MDA5.200 Caspase-12, genome, the EndoU gene might serve as an effective potential an inflammatory caspase, contributes to the innate response to therapeutic target for COVID-19 treatment. In addition, MAD5 can WNV infection by enhancing the K63-linked ubiquitination of RIG-I detect RNA from malaria parasites. Plasmodium infection induces by TRIM25.201 Furthermore, by associating with RIG-I and TRIM25, type I IFN in an MDA5- and MAVS-dependent manner and limits nuclear Ddf2-related 2 (NDR2), an NDR family serine/threonine the propagation of parasitemia.182,183 These observations suggest kinase, also positively regulates the addition of ubiquitin chains by that MDA5-mediated IFN production modulates innate immune TRIM25.202 However, the linear ubiquitin assembly complex responses to a wide range of pathogen infections. (LUBAC), consisting of the E3 heme-oxidized IRP2 ligase- 1L (HOIL-1L), and HOIL-1L-interacting protein (HOIP), negatively Interaction between MDA5 and endogenous RNA regulates RIG-I signaling by adding a K48-linked ubiquitin chain It is becoming clear that long dsRNAs, which are substrates of onto TRIM25, leading to its proteasomal degradation.203 This K48- MDA5, can be generated from endogenous transposable repeti- linked ubiquitin chain on TRIM25 is eliminated by a deubiquitinat- tive sequences, such as Alu and retroviral elements (ERVs).101,184 ing ubiquitin-specific peptidase (USP)15.204 A recent Alu repeats are present at more than one million sites in the study showed that NLRP12, a member of the NLR family, binds to human genome. Alu RNAs transcribed from inverted repeats in the TRIM25 and inhibits the TRIM25-mediated ubiquitination of RIG-I genome can be recognized by MDA5 when they form a dsRNA while simultaneously enhancing the degradation of RIG-I via K48- structure.185 However, in the steady state, A-to-I modification by linked ubiquitination of RIG-I by another E3 ligase, ring finger adenosine deaminase acting on RNA (ADAR)1 prevents the protein (RNF)125, demonstrating the functional interaction formation of immunostimulatory dsRNAs. ADAR1-deficient mice between the two PRR families.205 Notably, recent studies have are embryonic lethal due to global IFN induction;186 however, this reported that TRIM25 is capable of RNA binding, which is required phenotype is partially rescued by the deletion of MAVS or MDA5 for regulating signals by a mechanism that does not involve but not RIG-I,187,188 suggesting that ADAR1 is a specific suppressor ubiquitin ligase activity.206–209 of MDA5/MAVS signaling. A recent report showed that dysfunc- Riplet (also known as RNF135 and REUL) is another critical K63- tion caused by a mutation in the ADAR1 gene leads to the linked E3 ligase for RIG-I.210–212 Riplet conjugates ubiquitin on abnormal accumulation of Alu-derived dsRNA, resulting in the multiple Lys residues in the CARD and CTD of RIG-I. Although a overproduction of IFNs, which leads to autoimmune diseases, previous study suggested that Riplet functions upstream of including AGS.117,185,188–190 In addition, dsRNAs generated by the TRIM25 through the ubiquitination of Lys788 in the RIG-I CTD,82 activation of intrinsic ERVs following treatment with DNA more recent studies have shown that Riplet, but not TRIM25, may demethylating drugs or via the bidirectional transcription of be an essential regulator of RIG-I activation.213–215 Mechanistically, specific ERV regions can also be recognized by MDA5, leading to Cadena et al. proposed a model in which Riplet is involved in enhanced antitumor activity through IFN production.191–193 In upregulating RIG-I-mediated signaling via the initial ubiquitination adult human cells, tripartite motif (TRIM) protein 28 (TRIM28, also of oligomerized RIG-I on substrate dsRNAs and the subsequent known as KAP1) represses ERV transcription via epigenetic cross-bridging of RIG-I filaments on longer dsRNAs, leading to modification. However, the depletion of TRIM28 induces the signal amplification.214 Furthermore, Ube2D3, an E2 ligase, has RLR-mediated pathway, presumably as a result of dsRNA been identified as a coregulator of Riplet-mediated RIG-I activa- generation from ERV-derived RNAs.194 tion.213 However, another E3 ligase, zinc finger protein (ZNF)598, MDA5 can also recognize mitochondrial dsRNAs. The transloca- has been identified as a negative regulator of K63-linked tion of unstable mitochondrial dsRNAs from the mitochondria to ubiquitination by Riplet. ZNF598 conjugates the ubiquitin-like the cytoplasm is suppressed by the mitochondrial RNA helicase protein FAT10 (also known as ubiquitin D) on RIG-I to inhibit SUV3 and the polynucleotide phosphorylase PNPase; however, Riplet-mediated ubiquitination of RIG-I.216 loss of function of either protein induces cytoplasmic accumula- In addition to TRIM25 and Riplet, TRIM4 and MEX3C have also tion of dsRNAs, leading to the activation of MDA5-mediated IFN been identified by expression screening methods as positive production.195 The noncoding RNA ITPRIP-1 also plays an auxiliary regulatory E3 ligases of RIG-I.83,217 However, recent studies role in supporting MDA5 oligomer formation by binding to the C- suggest that these ligases are not involved in RIG-I-mediated terminus of MDA5.196 MAVS aggregation in vitro.213 The physiological function of the four E3 ligases, specifically TRIM25, Riplet, TRIM4 and MEX3C, in the activation of RIG-I remains unclear. This issue has been REGULATION BY HOST PROTEINS discussed in detail in recent excellent review articles.218,219 Briefly, Regulation by ubiquitin ligases and deubiquitinases although all of these molecules may be involved in the positive Posttranslational modification plays an essential role in regulating regulation of RIG-I activation, differing analytical conditions of RLR/MAVS-mediated signaling. In particular, regulation by ubiqui- recent reports has led to conflicting interpretations. Future tination has been extensively analyzed (Fig. 3d). In 2007, Gack analysis that takes into account differences in e viral, animal, et al. reported that Lys63 (K63)-linked ubiquitination at the Lys172 and cell types is expected to reveal the exact regulatory residue of RIG-I CARD by TRIM25, a RING-finger-containing E3 mechanisms based on specific usage and/or redundancy of the ubiquitin ligase, was required for CARD-mediated signaling molecules in RIG-I-mediated signal activation. activation.197 Subsequent analysis using an in vitro reconstitution Several E3 ligases that negatively regulate RIG-I have been assay system demonstrated that the unanchored K63-linked identified. For example, TRIM40 promotes proteasomal degrada- ubiquitin chain added by TRIM25 is also involved in RIG-I tion by conjugating K27- and K48-linked ubiquitin chains on both activation.198 The crystal structure of the RIG-I CARD with K63- RIG-I and MDA5,220 whereas RNF125 and RNF122 negatively linked ubiquitin revealed that both anchored and unanchored regulate RIG-I activation via K48-linked ubiquitination of RIG-I ubiquitin chains might participate in the stabilization of the “lock CARD.221,222 The RNF125-mediated degradation of RIG-I is washer”-like tetramer structure formed by RIG-I CARD.53 Moreover, antagonized by the SG component G3BP1 via enhanced self- TRIM25 regulates RIG-I-mediated signaling in coordination with ubiquitination of RNF125.89 After viral infection, the STUB1 E3 multiple host factors. Specifically, the mitochondrial-targeting ligase associates with the CARD of RIG-I via the trithorax group chaperone 14-3-3ε forms a complex with RIG-I and TRIM25 to protein mixed linage leukemia (MLL5), promoting K48-linked regulate the transition from the cytoplasm to MAM, which is ubiquitin-mediated degradation of RIG-I.223 Another E3 ligase, c- implicated in the RIG-I/MAVS interaction.199 ZCCHC3, a CCHC-type Cbl, is recruited, together with RIG-I, to Siglec-G, which is

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 546 expressed on the plasma membrane and promotes the Roles of other ubiquitin-like modifiers proteasomal degradation of RIG-I via K48-linked ubiquitin Regulation via ubiquitin-like modifications has also been demon- conjugation on the Lys813 residue of RIG-I.224 Moreover, Siglec- strated (Fig. 3e). An early study showed that RIG-I protein levels G belongs to the CLR family, suggesting an interaction between are negatively regulated by conjugation with the IFN-inducible different PRRs. ubiquitin-like protein ISG15 (ISGylation).249 Very recently, it has Regulation by several deubiquitinating has been been reported that ISGylation of MDA5 is essential for triggering demonstrated. USP4 and ovarian tumor-domain-containing ubi- IFN production in response to several RNA viruses, including quitin aldehyde-binding protein 1 (OTUB1) stabilize RIG-I proteins coronaviruses.250 Furthermore, TRIM38 positively regulates both by eliminating the K48-linked ubiquitin chains conjugated to RIG- RIG-I and MDA5 via SUMOylation at K43/K865 and K96/K888, I.225,226 Conversely, CYLD, a tumor suppressor gene expressed in respectively.251 The SUMOylation of RLRs inhibits degradation by cylindromatosis, negatively regulates RIG-I activation by deubi- K48-linked ubiquitination. However, in the late stage of infection, quitinating the K63-linked ubiquitin chains on RIG-I.227 Moreover, de-SUMOylation by SUMO-specific protease 2 (SENP2) enhances syndecan-4 (SDC4), which is a TM protein, complexes with both degradation, which leads to disrupted signaling. As mentioned RIG-I and CYLD to promote the CYLD-mediated deubiquitination earlier, Riplet-mediated RIG-I activation is attenuated by the of RIG-I, leading to subsequent signaling inhibition.228 Further- conjugation of FAT10 with RIG-I.216 Moreover, the noncovalent more, USP3,229 USP21,230 USP14,231 and USP27X232 also deubi- interaction between FAT10 and RIG-I suppresses RIG-I quitinate the K63-linked ubiquitin of RIG-I to inhibit RIG-I function. activation.252 An in vitro reconstitution analysis revealed that MDA5 CARD also requires K63-linked ubiquitin chains to activate the signaling Regulation by kinases and phosphatases pathway.233 However, the precise mechanism associated with RIG-I is negatively regulated by phosphorylation at Thr170 and ubiquitin-mediated regulation of MDA5 has remained unclear. Ser8 (Fig. 3f).253,254 Protein kinase C (PKC)-α and PKC-β are TRIM65 promotes K63-linked ubiquitination at Lys743 of MDA5 to involved in phosphorylation, which suppresses the TRIM25 enhance the oligomerization of MDA5 on dsRNAs.234 Moreover, interaction and K63-linked ubiquitination, resulting in the inhibi- arrestin domain-containing 4 (ARRDC4) interacts with MDA5 to tion of RIG-I-mediated signaling.255 However, as mentioned earlier, recruit TRIM65 in response to 71 (EV71) infection, the requirement of TRIM25 in RIG-I activation remains unclear and leading to the activation of MDA5-mediated signaling via Lys63- requires further analysis.213–215 Casein kinase 2 (CK2) phosphor- linked ubiquitination of MDA5.235 ylates RIG-I at Thr770, Ser854, and Ser855,256 which might be The regulation of MAVS by ubiquitin has also been extensively necessary for maintaining RIG-I in the autorepressed state. analyzed. K63-linked ubiquitination at Lys19, Lys311, and Lys461 Phosphorylation of RIG-I by DAPK1 at Thr667, located within the of MAVS by TRIM31 is critical for the formation of prion-like helicase domain, also suppresses RIG-I-mediated signaling. Since aggregates of MAVS.236 The scaffold protein FAS-associated factor DAPK1 activity is induced by RIG-I-mediated signaling, negative 1 (FAF1) negatively regulates TRIM31-mediated ubiquitination of feedback regulation by DAPK1 is suggested.257 However, MDA5 is MAVS by disrupting the interactions between MAVS and phosphorylated by RIO kinase 3 (RIOK3) at Ser828, which inhibits TRIM31.237 The phosphorylation of FAF1 by IKKε, which is MDA5 oligomerization.258 Moreover, phosphorylation at Ser88 in activated by RLR-mediated signaling, induces FAF1 degradation the MDA5 CARD also inhibits MDA5 activation.54 Importantly, and promotes the activation of MAVS-mediated signaling. OTUD4, dephosphorylation of RIG-I and MDA5 by PP1α and PP1γ induces a deubiquitinase, enhances MAVS-mediated signaling by stabiliz- the transition of RLRs to an activated state, indicating that ing MAVS and by eliminating K48-linked ubiquitins.238 dephosphorylation is one of the fundamental regulatory mechan- Several studies have demonstrated that the ubiquitination of isms for RLR activation.54 The regulation of MAVS by phosphor- MAVS negatively regulates MAVS-mediated signaling. In contrast ylation has also been reported. Protein kinase A (PKA) promotes to RIG-I, TRIM25 induces the degradation of MAVS via K48-linked the degradation of MAVS by MARCH5 by phosphorylating Thr54 ubiquitination at Lys7 and Lys10 of MAVS.239 The HECT domain- of MAVS and suppressing IFN induction.259 In contrast, phosphor- containing E3 ligase ITCH (also known as AIP4) binds to MAVS via ylation of MAVS Tyr by c-Abl positively regulates MAVS-mediated poly(C)-binding protein (PCBP) 1/2 and TAX1 binding protein IFN production.260,261 (TAX1BP1), which triggers K48-linked ubiquitination of MAVS and its subsequent degradation.240–242 Membrane-associated ring- Regulation by other modifications CH-type finger 5 (MARCH5), a mitochondrial ubiquitin ligase, and RIG-I is regulated by acetylation (Fig. 3e). For example, the RNF5 promote MAVS degradation via K48-linked ubiquitination deacetylation of the Lys858 and Lys909 residues of RIG-I via the at Lys7 and 500, as well as at Lys362 and 461, respectively. deacetylating enzyme HDAC6 is implicated in viral RNA detection Interestingly, ER-associated inactive rhomboid protein (iRhom) 2 by RIG-I;262,263 however, the molecular mechanisms underlying inhibits the MARCH5- and RNF5-mediated degradation of MAVS RIG-I acetylation remain unclear. Moreover, MAVS is positively and positively regulates MAVS-mediated signaling.243 The regulated by O-GlcNAcylation.264,265 In fact, Li et al. demonstrated TRIM29 protein conjugates with the K11-linked ubiquitin chain that O-GlcNAcylation at Ser366 of MAVS in response to VSV on MAVS for its degradation.244 However, MARCH8 is recruited by infection promotes K63-linked ubiquitination of MAVS by TRIM31, Tetherin (also known as BST2) to bind to MAVS and promote K27- leading to increased IFN production. linked ubiquitination at Lys7 of MAVS.245 Moreover, RNF34 conjugates with K27- and K29-linked ubiquitin chains at multiple Regulation by RNA-binding proteins Lys/Arg positions of MAVS. This K27/K29-linked ubiquitination Since RLRs are RNA-binding proteins, activation of RLR-mediated induces NDP52/CALCOCO2-dependent autophagic degradation signaling is regulated by the coordination of various host RNA- of MAVS.246 The activity of MAVS is also regulated by binding proteins (Fig. 3g). Members of the 2′-5′ oligoadenylate (2- deubiquitinases, including YOD1,247 and OTUD3,248 which 5 A) synthetase (OAS) family, which are classical antiviral ISGs, are repress MAVS-mediated signaling by removing the K63-linked activated by viral dsRNA and eliminate viral RNA via the activation ubiquitin chains. Although a considerable number of studies of the host ribonuclease RNase L.266 Interestingly, human OAS-like have reported the regulation of RLR/MAVS-mediated signaling protein (OASL) is a member of the OAS family that contains via ubiquitination/deubiquitination, the mechanisms underlying a unique ubiquitin-like domain but lacks oligoadenylation these regulatory processes remain unclear, and further studies activity. The ubiquitin-like domain of OASL mimics the K63- are warranted. linked polyubiquitin moiety and induces the oligomerization

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 547 of RIG-I on RNA substrates, thereby enhancing RIG-I-mediated and MAVS.296 SIDT2, the mammalian ortholog of the Caenorhab- signaling.267 ditis elegans dsRNA transporter systemic RNA interference PKR, another classic antiviral ISG, has been extensively defective protein 1 (SID-1), has been shown to enhance RLR analyzed.268 PKR inhibits viral growth by potently repressing host signaling by enhancing the transport of internalized dsRNA from translation through the phosphorylation of eIF-2α. In terms of the endosomal compartments to the cytoplasm for viral detection by regulatory function of RLRs, as mentioned earlier, SGs formed via RLRs.297 PKR mediation might play critical roles as platforms for viral RNA RLR/MAVS-mediated signaling is inhibited by multiple host detection by RLRs, suggesting an indirect regulatory function of factors. Soonthornvacharin et al. identified K-homology splicing PKR in RLR-mediated signaling.269,270 Moreover, the RNA helicase regulatory protein (KHSRP) as a negative regulatory molecule for DHX36 facilitates PKR-dependent SG formation, resulting in RIG-I-mediated signaling by genome-wide RNAi screening.298 increased RLR-mediated IFN production.75 KHSRP suppresses the early activation of RIG-I by directly Protein activators of PKR (PACT) and TRBP are dsRNA-binding associating with RIG-I CTD. The predicted methyltransferase proteins that exert positive and negative regulatory functions of TTLL12, which was identified as a host regulator that inhibits PKR, respectively.271 Initially, PACT was reported to participate in SeV-induced RIG-I-mediated signaling, binds to MAVS to suppress IFN production272 but was subsequently shown to also physically signaling.299 DNAJ homolog subfamily B member 1 (DNAJB1), associate with both RIG-I and MDA5, effectively augmenting their which is a heat protein (HSP) 40 family member protein, was signaling by enhancing ATPase function and oligomerization, identified as an MDA5-interacting molecule.87 In response to viral respectively.273,274 Moreover, recent studies have indicated that infection, DNAJB1 is translocated with HSP70 to SGs, where the PACT-LGP2 interaction is required for upregulated MDA5- it inhibits the formation of signal-competent MDA5/MAVS mediated signaling.275,276 However, the regulation of RLR- filaments. The zinc finger FYVE domain-containing protein mediated signaling by PACT is antagonized by viral factors, ZFYVE1 specifically associates with MDA5 and inhibits MDA5- including VP35 of virus (EBOV), NS1 of IAV, and NP of mediated signaling.300 FK506-binding protein 8 (FKBP8) inhibits .277–279 Importantly, the 4a protein of Middle East the RLR-MAVS pathway by directly binding to MAVS.301 Eckard respiratory syndrome coronavirus (MERS-CoV) and the nucleocap- et al. demonstrated that SKIV2L, which is a cytosolic 3′-5′ RNA sid (N) protein of MHV and SARS-CoV directly interact with PACT to exosome, prevents the constitutive activation of RLRs via the inhibit RLR-mediated signaling,280,281 suggesting that it is a degradation of endogenous RNAs, which are produced by inositol possible therapeutic target for COVID-19. In addition to the requiring enzyme-1 (IRE-1)-mediated cytoplasmic splicing in inhibitory function of PKR, TRBP enhances MDA5-mediated innate response to the unfolded protein response.302 They also observed responses by physically interacting with LGP2 in response to that patients with human trichohepatoenteric syndrome (THES), infection by , which belongs to the Picornaviridae caused by a SKIV2L loss-of-function mutation, exhibit an excessive family.282 However, TRBP plays an essential role in miRNA IFN signature, indicating that the metabolic machinery of processing from precursor RNAs in conjunction with the endogenous RLR substrates consists of exosomal RNAs.303 NLRX1, ribonuclease Dicer.283 Recent studies have shown that the a mitochondrial-localized NLR family member, suppresses RIG-I- enhanced expression of LGP2 in response to viral infection mediated signaling by interrupting RIG-I and MAVS.304,305 A suppresses Dicer-mediated miRNA maturation via the LGP2- subsequent study demonstrated that NLRX1 not only suppresses induced sequestration of TRBP from the Dicer-TRBP complex.34 RIG-I-mediated signaling but also promotes autophagosome Genome-wide analysis revealed that the suppression of RNA formation in association with mitochondrial Tu translation silencing by TRBP-dependent miRNA leads to enhanced specific elongation factor (TUFM), leading to enhanced VSV replication.306 gene expression, including cell death-related gene expression, The involvement of autophagy and related proteins, such as Atg5- suggesting a potential novel antiviral defense mechanism induced Atg12, in RLR-mediated signaling has been demonstrated by the LGP2-TRBP interaction.35 (Fig. 3i).307,308 Recently, leucine-rich repeat-containing protein 25 The involvement of multiple RNA helicases in RLR-mediated (LRRC25) has been reported to interact with ISGylated RIG-I and to signaling has been demonstrated. For example, DDX60 can promote the degradation of RIG-I in autophagosomes to suppress enhance RIG-I-mediated signaling by associating with RIG-I, IFN production.309 However, LRRC59 inhibits the degradation of indicating its role as a sentinel RNA sensor.284,285 However, ISGylated RIG-I by competing with LRRC25.310 Coiled-coil domain- another report revealed that DDX60 deficiency does not affect IFN containing 50 (CCDC50), a novel autophagy receptor, also induces production in response to several RNA viruses or dsRNAs, the degradation of RIG-I/MDA5 by recruiting it as an autophagic suggesting that it might function in antiviral responses against cargo in a K63-linked ubiquitin-dependent manner to inhibit the very specific viral species.286 In addition, DDX6, DHX29, and IFN pathway.311 Immunity-related GTPase M (IRGM), in which loss- DHX15 can also function as cosensors of RIG-I and MDA5 by of-function mutations are known to be genetically associated with directly interacting with RLRs.287–290 However, DDX3, which was various immune diseases,312 binds to RIG-I/MAVS to promote initially identified as a MAVS-interacting helicase via two-hybrid autophagic degradation.313 Finally, a recent study revealed that screening,291 participates in the detection of aberrant human caspase-3 negatively regulates this signaling pathway via the immunodeficiency virus type 1 (HIV-1) RNA in dendritic cells (DCs), degradation of MAVS and IRF-3.314 which induces MAVS-mediated signaling independent of RLRs.292 Furthermore, DHX9 is also involved in the RLR-independent activation of MAVS-mediated signaling in response to dsRNA in REGULATION BY VIRAL PROTEINS DCs.293 Since the molecular mechanisms of these observations Most viruses have evolved strategies that favor their proliferation remain unclear, further analysis is necessary. by suppressing the host innate immune system.315 In particular, the target molecules range from RLR/MAVS to downstream Regulation by other host proteins signaling molecules such as TRAFs, IKKs, IRFs, NF-κBs, IFNRs, JAKs, 14-3-3 proteins, including 14-3-3ε and 14-3-3η, can function as and STATs. In this section, we summarize recent findings mitochondrial transporting chaperones for RIG-I and MDA5, regarding direct interactions between RLR/MAVS and pathogenic respectively (Fig. 3h).199,294 NS3 proteins of DENV, ZIKV, and viruses (Fig. 3). presumably WNV antagonize RLR-mediated signaling by seques- trating 14-3-3 proteins.295 Zyxin, which is involved in actin Negative-sense ssRNA viruses polymerization, localizes with MAVS on the mitochondria and RNA viruses with single-stranded, negative-sense inhibit functions as a scaffold protein for the interaction between RLRs RLR/MAVS in various ways. For example, the V protein of viruses

Cellular & Molecular Immunology (2021) 18:539 – 555 Regulation of RIG-I-like receptor-mediated signaling: interaction between. . . K Onomoto et al. 548 belonging to the family suppresses IFN produc- infected cells.342 NS4A of DENV and ZIKV inhibits IFN signaling tion by interacting with MDA5 and LGP2 but not with RIG-I.316–318 by directly interacting with MAVS.343,344 produce Moreover, the V proteins of virus (MV) and unique subgenomic flavivirus RNA (sfRNA), a noncoding RNA (NiV) inhibit dephosphorylation-mediated activation of MDA5 by derived from the 3′ UTR, which is associated with viral directly associating with PP1α/γ.319 However, since paramyxo- pathogenicity.345 A comparative analysis of the DENV viral clade viruses have a 5′-triphosphate moiety, which is a non-self-RNA revealed that an increase in sfRNA inhibits the deubiquitination of signature specific for RIG-I, the biological significance of the V- the K48-linked ubiquitin chains of TRIM25, which suppresses the mediated repression of MDA5 is unclear. Notably, MDA5 has been RIG-I-mediated signaling induced by TRIM25.206 suggested to be involved in SeV-induced IFN production in the Viruses belonging to the Picornaviridae family terminate innate late stages of infection; thus, the mechanism of action of V immune signaling using their protease activity, which is essential proteins might differ depending on the time points of the antiviral for processing viral polyproteins. have two pro- response.320 The V proteins of NiV, SeV, and parainfluenzavirus teases, 2A and 3C. EV71, coxsackievirus 3 (CBV3), and poliovirus (PIV) can suppress RIG-I-mediated signaling by preventing TRIM25- (PV) degrade MDA5 via their respective 2A protease.346 Moreover, mediated ubiquitin conjugation on RIG-I via complex formation MAVS can also be degraded by 2A and 3C proteases.346–349 In the with both RIG-I and TRIM25.321 However, the V proteins of several case of PV infection, degradation of MDA5 by the host proteasome paramyxoviruses, including NDV, suppress IFN signaling by and caspase has been suggested.350 Furthermore, RIG-I can also enhancing MAVS degradation by the RNF5 E3 ligase.322 be processed by enteroviral proteases; however, its biological Moreover, the NS1 protein of human respiratory syncytial virus significance is unclear.351 In addition, 3C of PV and EMCV inhibits (RSV), a member of the Pneumoviridae family, also interacts with SG formation by degrading G3BP1 (an essential regulator of TRIM25 and inhibits ubiquitin-conjugation of RIG-I.323 In addition, SGs)352 and represses SG-mediated enhancement of RLR- the NS2 protein of RSV directly binds to RIG-I CARD, which inhibits mediated signaling.353 Foot-and-mouth disease virus (FMDV), an the association between activated RIG-I and MAVS.324 The VP35 member, encodes leader protease (Lpro), a protein of EBOV and Marburgvirus (MARV), which belongs to the unique protein that degrades LGP2 and indirectly suppresses family, can bind dsRNA and antagonize RLR-mediated MDA5-mediated signaling.354 A recent study indicated that Lpro signaling.325,326 In addition, VP35 can suppress RIG-I-mediated inhibits signaling by degrading MAVS and TBK1.355 However, Lpro signaling by suppressing RIG-I-PACT interactions.277 BDV, a has been shown to deubiquitinate RIG-I, thereby terminating RIG- member of the Bornaviridae family, has a strategy to trim several I-mediated signaling.356 Furthermore, the 2B protein of FMDV nucleotides at the 5′ end of the viral genome to evade recognition specifically inhibits RIG-I;357 however, it remains unclear why these by RIG-I, resulting in latent infection.147,327 proteins target RIG-I. Viruses belonging to the family, including IAV, Viruses belonging to the Bunyavirales order replicate their also have negative-sense ssRNAs. The IAV NS1 protein, derived genomic RNA using a “prime-and-realign” machinery.358 The from the smallest viral genome of the eight segments that make genomic RNA of CCHFV () and HTNV () up the IAV genome, is a well-known multifunctional virulence avoids RIG-I recognition upon the removal of the triphosphate factor. NS1 contains an N-terminal dsRNA-binding domain and C- moiety at the 5′ end of the single G overhang and generating a 5′- terminal effector domain that suppress antiviral responses monophosphate by “prime-and-realign” replication.147 However, through distinct actions.328 While the N-terminal dsRNA-binding viruses belonging to the Arenaviridae family have been reported domain antagonistically represses RIG-I-mediated IFN to escape RIG-I detection precisely because of its 5′ overhang production,132,329 NS1 also inhibits RIG-I via direct interaction in structure, even though the 5′-triphosphate G is not removed.359 an RNA-independent manner.330 NS1 also associates with TRIM25 Moreover, the Z protein of Arenaviridae viruses such as Junin virus and Riplet and represses the K63-linked ubiquitination of RIG- (JUNV) suppresses the IFN response via direct interaction with RIG- I.331,332 The C-terminal effector domain of NS1 interacts with I.360 This suppression mechanism is common among pathogenic several host factors, including PKR.333 IAVs that lack NS1 can , suggesting that it is an essential factor for their efficiently activate PKR and enhance RIG-I-mediated IFN produc- pathogenicity. However, the NS protein of severe fever with tion via SG formation.73 The PB1-F2 protein is a mitochondrial- syndrome virus (SFTSV), a family member of localized peptide (87 amino acids) translated from the PB1- the family, associates with RIG-I and TRIM25 and encoding RNA segment and contributes to virulence.334 Although suppresses RIG-I-mediated signaling by repressing the K63-linked PB1-F2 inhibits MAVS-mediated signaling via direct interaction,335 ubiquitination of RIG-I.361,362 Although inhibition of RIG-I by its PB1-F2 of H7N9 highly pathogenic IAVs inhibits K63-linked direction interaction with the NS protein of Toscana virus (TOSV) ubiquitination of MAVS by suppressing the TRIM31-MAVS inter- has been suggested,363 a recent report revealed that NS can action.336 PB1-F2 can also repress MAVS-mediated signaling by function as an E3 ubiquitin ligase and degrade the RIG-I protein to degrading MAVS via mitophagy.337 However, M2, a membrane terminate signaling.364 protein generated from the M-encoding RNA segment, enhances Finally, we describe recent findings on the interaction between MAVS-mediated signaling by antagonizing reactive oxygen Coronaviridae family members and RLR/MAVS. Pathogenic human species (ROS)-induced autophagy activation and increasing the coronaviruses, including SARS-CoV, SARS-CoV-2, and MERS-CoV, number of MAVS aggregates on the mitochondria.338 are classified as betacoronaviruses. Since the production of IFNs is significantly attenuated in cells infected with these viruses,365–367 Positive-sense ssRNA viruses suppression of the RLR/MAVS pathway by viral proteins might be HCV, a member of the Flaviviridae family, encodes protease NS3/ a critical therapeutic target for coronaviral infectious diseases, 4A, which is essential for the cleavage of the viral polyprotein. including COVID-19. ORF9 of SARS-CoV, which encodes the N NS3/4A inhibits IFN production via the degradation of MAVS.339,340 protein, interacts with TRIM25 and inhibits RIG-I via the NS3/4A also disrupts RIG-I-mediated signaling by degrading ubiquitination of RIG-I.368 SARS-CoV ORF-9b, a short peptide with Riplet.82 Since many flaviviruses are transmitted by arthropods 98 amino acids translated based on a discordant ORF in the N (known as ), they share several standard features. The gene, localizes to the mitochondria, altering their morphology by phosphomimetic amino acid sequences in the NS3 proteins of inhibiting mitochondrial fission and thereby promoting the ZIKV, DENV, and WNV repress RLR-mediated signaling by PCBP2-mediated degradation of MAVS by the E3 ubiquitin ligase associating with the scaffold proteins 14-3-3ε and η.295,341 The ITCH/AIP4.369 In addition, MERS-CoV is closely related to SARS- proteins of DENV and WNV suppress IFN-λ gene expression CoVs; however, the genomic structures differ slightly. Through its via the degradation of peroxisome-localized MAVS in virus- dsRNA-binding activity, the unique 4a protein of MERS-CoV

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